Neurotransmitters are released in a very fast reaction (<1ms) acutely triggered by Ca2+ Neurotransmitter release is also regulated by Ca2+ at multiple levels in processes of synaptic plasticity that may underlie learning and memory formation. Characterization of the molecular basis of neurotransmitter release and its Ca2+ regulation is thus crucial to understand brain function and would greatly facilitate the development of therapies for neurological diseases with a presynaptic origin. The observation that a variety of proteins implicated in neurotransmitter release contain a universal Ca2+-binding module known as the C2-domain suggests that C2-domains play multiple roles in Ca2+ regulation of neurotransmitter release. These proteins include synaptotagmin, rabphilin, DOC2s, munc13, Rim, aczonin and intersectin. Synaptotagmin, a synaptic vesicle protein that contains two C2-domains in its cytoplasmic region, is the most extensively studied among these proteins because of its putative role as the Ca2+ sensor that triggers release. However, the function of synaptotagmin remains largely unclear. To shed light on this function, an integrated approach involving structural, biochemical and genetic experiments is proposed. The approach is intended to characterize structural features of synaptotagmin that may be important for its function and for interactions with essential components of the synaptic exocytotic machinery, and to test the importance of these interactions in vivo. In addition, the research proposed is intended to extend the studies on synaptotagmin to other C2-domain proteins that have been implicated in neurotransmitter release but whose functions are also unclear. The primary goals in this area are to have a comparative picture of the Ca2+ and phospholipid binding properties of presynaptic C2-domains, to understand the structural determinants of these properties, and to determine whether the most interesting among these C2-domains have structural features that may underlie their functions. To achieve these goals, the five specific aims proposed focus on the following areas. 1. Structure and Ca2+-binding properties of the cytoplasmic region of synaptotagmin. 2. Structural basis of protein-protein interactions involving synaptotagmin. 3. Mutational analysis of synaptotagmin function in vivo. 4. Ca2+ and phospholipid binding to C2-domains. 5. Three dimensional structures of the C2-domains from munc13, RIM and aczonin. Overall, this research will yield critical information to understand the function of synaptotagmin and will reveal the structural determinants for Ca2+ and phospholipid binding to C2-domains in general. Characterization of these properties will not only yield insights into the functions of C2-domains in neurotransmitter release but will also facilitate future studies that will further clarify these functions.